| Tristeza caused by Citrus tristeza virus (CTV) is one of the most important diseases to the citrus industry, and has caused huge economic loss throughout the world. With the structure adjustment of the citrus varieties in China, the cultivation proportion of pomelo and sweet orange had been promoted, the damage caused by stem pitting isolates of CTV has been becoming more severe. Both domestic and overseas reasearch data showed that mild strain cross protection (MSCP) would be the most effective management against stem pitting in areas where virulent CTV isolates and Toxoptera citricida (Kirkaldy) were commonly distributed. Due to the genotype composition of severe and mild strains of CTV, associated with the control effect of mild strain, quantitative dectection of the different CTV genotypes of the mild strain would help further study on the mechanism of MSCP.In this study, a pair of primers T3-4F/R were designed within the ORFla of T3 genotype, and the SYBR Green I real-time RT-PCR detection system was established with optimized reaction condition. And then, its sensitivity and reproducibility were estimated. A rapid specific SYBR Green I RT-PCR method for detecting of T3 genotype was established, and used to monitor the different genotype content of the mild strains CT11 and CT31 in field, together with reported real-time fluorescence quantitative methods, detecting T30, T36, VT genotypes. In the main while, the effect of MSCP was evalued in the field with mild strains CT11 and CT31 pre-inoculated. The following results have been achieved:1. The SYBR Green I RT-qPCR detection system was established with optimized reaction condition.This system had strong specificity and high sensitivity to T3 genotype CTV, which was 100 times higher than that of conventional RT-PCR. A good linear correlation (R2=0.992) obtained from two standard curve of cRNA. The amplification efficiency was 97.1%. Three-time repeats revealed that the coefficients of variation between the intra-and inter-assay were both within 2.94%. It could stably detect the T3 genotype CTV in the field.2. Four genotypes of CTV within the samples inoculated with mild CT11, CT31 respectively, were detected using RT-qPCR detecton. Results of CTV temporal and spatial distribution showed that contents of all detected genotypes varied within different tissues along with time, and contents in summer shoots were higher than that in spring pins and autumn growths. The content of T30 genotype CTV seemed to be the highest of 4 different genotypes.3. MSCP management was evaluated with analysis of growth condition of the tree, fruit quality and content of T30 genotype CTV. The results show that those samples inoculated with mild CT31 and CT11, were well protected from virulent CTV in field, and samples with CT31 seemed to be much better than those with CT11. The proportion of average internode length more than 20mm branches in mild CT31, CT11 and controlled plant was 36%,20% and 16% respectively. The proportion of no stem-pitting symptoms in mild CT31, CT11 and controlled plant was 92%,64% and 46%respectively. The proportion of the big fruit in mild CT31, CT11 and controlled plant was 85.7%,68.7% and 32.2% respectively. Besides that, it was also noted that the severity of stem-pitting symptoms exhibitted some linkage to the content of T30 genotype CTV. The stem-pitting was observed to be much milder, or ever no obvious syptoms when the contents of T30 genotype was detected to be higher, and vice versa, lower content of T30 led to more severe stem-pitting. |
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